Se-E and Te-E were obtained with CA/Pr-E and Se-E/CHI of 70/30 and 50/50% (w/w), respectively. The Se-E was the most stable compared to the Pr-E and Te-E when subjected to different pHs; nevertheless, once the NaCl concentration rose, no variations in the ζ-potential of all emulsions studied or destabilization were observed. The Se-E and Te-E derived provided higher astaxanthin retention (>95%) during the spray-drying process compared to Pr-E (around 88%). The results indicated that these astaxanthin multilayer emulsions show considerable potential as a functional ingredient in food products.The utilization of astaxanthin in food processing is considered to be narrow because of its substandard solubility in aqueous matrices and the instability of chemical compounds during the processing of food and the instability of chemical compounds during the processing of food. The investigation sought to evaluate multilayer emulsions stabilized by ionic interfacial layers of lupin protein isolate (LPI), ι-carrageenan (CA), and chitosan (CHI) on the physical stability of the emulsion as well as the retention of astaxanthin during the spray drying process. Primary emulsion (Pr-E) was prepared by adding LPI on oil droplet surfaces containing astaxanthin. The homogenization pressure and cycles to obtain the Pr-E were investigated. The secondary emulsion (Se-E) and tertiary emulsion (Te-E) were elaborated by mixing CA/Pr-E and CHI/Se-E, respectively. Emulsion stability was assessed under different environmental stresses (pH and NaCl). Astaxanthin retention of emulsions was determined immediately after finishing the spray-drying process. The results showed that Pr-E was stabilized with 1.0% ( There is a widespread use of this technology in the industry of food due to the relatively low cost and short drying timeframe in comparison with the freeze-drying method . Since sechnique .Currently, some studies indicate that the application of vegetable proteins and ionic polysaccharides used to produce multi-layer o/w emulsions based on interfacial engineering technology enables the creation of natural supply systems of improved stability . In multIn this study, lupin protein isolate (LPI) was used to stabilize and formulate the first layer of Pr-E, given its notable emulsifying qualities, which were determined previously ,15. FollCA is a linear sulfated anionic polysaccharide extracted from red algae (Rhodophyceae), typically applied in the food industry for gelling and thickening applications . An advaAstaxanthin is a xanthophyll carotenoid that is present in several microorganisms and marine wildlife. The natural sources of astaxanthin are microorganisms, phytoplankton, marine species, and seafood for instance shrimp, lobster, asteroidean, algae, fish, crustacean, trout, krill, red sea bream, and salmon . NowadayConsequently, the study aimed to evaluate the effect of multilayer emulsions stabilized by ionic interfacial layers of LPI, CA, and CHI on the physical stability of the emulsion and the retention of astaxanthin during the spray-drying process. Multilayer emulsions were produced by sequentially adding CA and CHI to surfaces of LPI-coated oil droplets. The results obtained through this study will allow a greater understanding of the role of multilayer emulsions with respect to the protection of astaxanthin for potential applications in the food powder industry.Lupinus luteus) was provided by Agriaquaculture Nutritional Genomic Center. The ι-carrageenan was bought from Sigma Aldrich . Chitosan used in the study was obtained from Xi’an Surnature Biological Technology Co. Ltd. . Sunflower oil was bought in a local market  and astaxanthin oleoresin of Haematococcus pluvialis was supplied by Atacama Bio Natural Products S.A. . Maltodextrin  was bought from Sigma Aldrich .LPI from yellow lupin seeds (w/v) has been determined by an optical tensiometer model 250-F4  at ambient temperature. To measure IT, the “pendant drop” (a drop of LPI solution dispersed in oil) method was employed. It entails taking a picture of the dispersion droplet using a high-speed digital camera and examining its measurements. An axisymmetric drop (8 μL) of LPI dispersion was provided and placed at the tip of the needle inside a quartz recipient with 30 mL of oil phase over a period of time to cause the adsorption of the emulsifier at the oil-water interface. The interfacial tension (mN/m) was measured using the DropImage software  by adjusting the Laplace equation to the shape of the drop. The measurement of each sample was performed in triplicate.The IT at the o/w interface for different concentrations of LPI (from 0.1 to 2.0% w/w) astaxanthin and 90% (w/w) sunflower oil. The Pr-E was obtained by mixing a 10% (w/w) oil phase with 90% (w/w) protein emulsifier (from 0.1 to 2.0% w/v of LPI) at pH 3. The mixture was homogenized using a high-speed blender  at 10,000 rpm for 5 min, and afterward, it was blended in a high-pressure homogenizer (HPH) . The impact of HPH process conditions on the size of particles and the polydispersity index of Pr-E was determined. Different homogenization pressures (10 to 70 MPa) and the number of cycles (1 to 5) were analyzed. The oil phase was prepared with a 10%  with Pr-E (CA/Pr-E) at different proportions . Then, tertiary emulsion (Te-E) was produced with a mixture of solutions of CHI (0.1–0.4% w/v at pH 3) and Se-E (CHI/Se-E) at different proportions . The homogenization of Se-E and Te-E was carried out at 5000 rpm for 2 min each one, after which two cycles at 15 MPa were carried out using an HPH. After that, the emulsions were homogenized at room temperature for 1 h with a magnetic stirrer.The multilayer emulsions were elaborated in the following manner: the secondary emulsion (Se-E) was elaborated with a mixture of CA solutions (0.1–0.4% v/v) in order to prevent multiple dispersion effects. The PdI value refers to a measurement of the particle size distribution. Analyses were conducted in triplicate.The mean size of particles and Polydispersity Index (PdI) of the emulsions were estimated through the dynamic light scattering in a laser diffractometer . The emulsions were diluted before the examination with milli-Q water at a proportion of 1:100 (v/v), then placed in zeta cells made of plastic  and equilibrated at 25 °C during 60 s. These examinations were carried out three times.The ζ-potential of the emulsions was examined through an instrument combining dynamic light scattering and electrophoresis . Therefore, the emulsions were dissolved with milli-Q water before analysis in a proportion of 1:100  to determine the size of the drops and the state of aggregation of oil drops in each emulsion. Therefore, a drop of the emulsion was put on the microscope slide with a coverslip. Images of the samples were obtained with software to process digital images .e represents the total height of the emulsion (mm) and Hs refers to the height of the cream layer (mm), which was visually determined in function of time.The stability of the emulsion regarding creaming was analyzed with the Creaming Index (CI) method . For thiThe effect of different pH values (3–7) was tested on the properties of the emulsions. The pH was modified with the addition of an HCl or NaOH solution. Also, the emulsions with a difference in NaCl concentration (0–300 mM) were elaborated blending the initial emulsions with different proportions of salt and buffer solution. The emulsions prepared were stored under ambient temperature for 24 h before the analysis of the ζ-potential.The emulsions were placed in a B-290 mini-spray dryer . Previous to spray drying, maltodextrin was added to the emulsions to obtain a total solids content of 15% and then these mixtures were stirred for 1 h. The spray dryer was used at an inlet air temperature of 120 and 160 °C, 4 mL/min of feeding rate and 85% of aspiration rate. The microcapsules were stored at −20 °C for later characterization.v/v) and vortexed for 1 min. The organic phase was isolated/detached by centrifugation at 1000× g for 5 min. Astaxanthin was assayed in the supernatant by Ultraviolet–visible (UV-Vis) spectrophotometry  at 478 nm with a microplate reader. Pure hexane was utilized as a blank in all determinations. The astaxanthin content was estimated, by applying a calibration curve using Equation (2):sm stands for absorbance of the sample, Wsm stands for the mass of the sample in grams and V represents the dilution volume (mL).The astaxanthin content of emulsions was assessed after the spray-drying process in accordance with the methodology defined by Burgos-Diaz et al. . In the 1 represents the theoretical content of astaxanthin in the powder emulsions (8.5 mg Astax/g emulsion), and C2 indicates the content of astaxanthin after finishing the spray-drying process.The retention of encapsulated astaxanthin was determined immediately after finishing the spray-drying process . The astThe results shown represent the average and the standard deviation estimated according to these replicate measurements. An analysis of variance (ANOVA) was conducted using a significance level fixed at 0.05. The Duncan test was conducted If relevant disparities were identified using the ANOVA. The statistical analysis was conducted using InfoStat software.Interfacial properties of proteins are essential in order for o/w emulsions to be formed and stabilized . On thisp < 0.05) when the time of protein adsorption at the oil-water interface increased from 0 to 800 s. The function of an emulsifier like proteins is to lower the interfacial tension covering the surface of the new-formed oil droplets with a protective layer and to stabilize the droplets in the continuous aqueous phase [p > 0.05) when time increased from 800 to 3600 s. These results showed that the period of LPI saturation at the oil-water interface is from 800 s onwards, given that relatively constant interfacial tension values were achieved. During this phase, the protein was located at the oil-water interface, where it could adopt a new structural configuration in which polar groups interact with water, and non-polar groups interact with oil, a process known as interfacial denaturation [us phase . Accordius phase , the IT turation .w/v) the decrease in IT in function of time was not considerably different (p > 0.05); however, IT as function of time was substantially reduced (p < 0.05) as the LPI concentration rose from 0.1 to 1.0% (w/v). This decrease in IT as LPI concentration increases could be associated with faster absorption of proteins on the oil droplet surface [p > 0.05) when there was an increase of LPI concentration from 1.0 to 2.0% (w/v). Therefore, it was demonstrated that 1.0% of LPI would be sufficient to saturate the oil-water interface of the Pr-E. On the other hand, the results have shown that different concentrations of LPI can be adsorbed by the oil/water interface, as demonstrated by the reduction in the IT of the same interface when proteins are absent (0% LPI). Between LPI concentrations of 0.1 and 0.5%  of the oil phase with 90% (w/w) of the emulsifier solution containing 1% (w/v) of LPI. The results revealed that particle size (p > 0.05) when homogenization pressure increased from 10 to 30 MPa. However, particle size PdI was reduced considerably (p < 0.05) when homogenization pressure rose from 30 to 50 MPa. Moreover, there was no relevant difference observed (p > 0.05) when the homogenization pressure enhanced from 50 to 70 MPa. The emulsion was prepared by adding 10% (cle size a and PdIcle size b of oil p < 0.05) when the number of homogenization cycles increased from one to three cycles for all homogenization pressures. Despite that, the increase in the number of homogenization cycles from three to five did not significantly affect (p > 0.05) the particle size and PdI. Therefore, the homogenization process conditions selected to prepare the Pr-E were 50 MPa and three cycles, resulting in a particle size of 0.87 ± 0.07 µm. At the same time, the PdI value of 0.583 ± 0.05 suggests partial polydispersity of oil droplets in the emulsion. Similar behavior was reported in previous studies with homogenization pressures (from 30 to 100 MPa) on the particle size (below 1 μm) in the o/w emulsions prepared with cod protein isolate [Particle size and PdI were reduced substantially  show, predominantly, great stability because of strong repulsive electrostatic forces between the droplets .The addition of CA on Pr-E (CA/Pr-E) and CHI on Se-E (CHI/Se-E) needed in different ratios to fully coat the oil droplets of the emulsion was assessed . More spp < 0.05) when the addition of CA on Pr-E increased. The oil droplet ζ-potential value was increased considerably (p < 0.05) when the addition of CA/ Pr-E increased from 50/50 to 60/40% (w/w). Furthermore, between 60/40 and 80/20% (w/w), no considerable difference (p > 0.05) were perceived regarding the negative electrical charge, indicating that a constant ζ-potential value was obtained from 60/40% (w/w). However, some oil droplet aggregations were visualized (data not shown) in the sample tube of emulsions formed with 60/40% (w/w) unlike the emulsions obtained between 70/30 and 80/20% (w/w). Presumably, in the 60/40% (w/w) ratio, there were not enough CA anionic groups to be completely adsorbed on the surface of the LPI-stabilized oil droplets. Therefore, 70/30% (w/w) of CA/Pr-E was selected to prepare the Se-E. Meanwhile, the ζ-potential of the oil droplets showed an increase when the CA content was augmented from 0.1 to 0.4% (w/v), thus reaching a constant value around −54 mV. This implies that oil droplet surfaces were saturated completely by the CA molecules Burgos-Díaz et al. [p > 0.05) when the addition of CHI in Se-E was increased from 50/50 to 80/20% (w/w), thereby attaining a constant value around 50.4 mV with 50/50% (w/w). This effect is like the adsorption of cationic CHI molecules on the surfaces of the Se-E oil droplets given the strong electrostatic force of attraction between them. As several authors have reported, colloidal systems presenting high ζ-potential values, greater than +25 mV or less than −25 mV, show, predominantly, great stability because of strong repulsive electrostatic forces between the droplets [z et al.  points odroplets ,34. It wThe physical stability of Pr-E, Se-E, and Te-E was examined based on the CI during storage for 7 days . The formation of cream is a result of differences in density between the dispersed phase and the dispersion medium. The CI (%) consists of the separation of two phases in the emulsion, characterized by the formation of a cream layer in the upper part of the sample tube and a clarification zone in the lower part of the tube . The resThe optical microscopy images  revealedIn Emulsions constitute systems that are thermodynamically unstable, which can be affected by environmental conditions commonly found during food processing like pH and salt concentration . ConsequThe experiment aimed to analyze the effect of pH on emulsion stability a. ChangePr-E ζ-potential varied from 33.1 ± 3.89 mV to −32.2 ± 3.25 mV when the pH increased from 3 to 7, because of the amphoteric nature of proteins ; at thisThe Se-E showed that the ζ-potential value changed from −50.9 ± 1.20 mV at pH 3 to −58.4 ± 1.70 mV at pH 7. The Se-E stabilized by CA and LPI showed negatively charged ζ-potential values over the entire range of pH values, which led to a high electrostatic repulsion among the oil droplets covered with CA/LPI layers, enhancing the physical stability (without phase separation) of the Se-E in comparison to a Pr-E. Similar behavior was observed by Gu et al.  when sec3+ groups [The Te-E presented ζ-potential values of positive charge from 51.5 ± 1.41 mV at pH 3 to 4.3 ± 1.20 mV at pH 7. However, a decrease in the ζ-potential was observed at pH values over 4.3, causing destabilization (emulsion aggregation) of the Te-E. This charge loss has been attributed previously to the proximity to the chitosan pKa (6.5) causing neutralization of NH+ groups . Such be+ groups , who evap < 0.05) when compared to Pr-E, showing the protective effect of multilayer emulsions. Also, no relevant differences were observed (p > 0.05) in the retention of astaxanthin between Se-E and Te-E. In addition, after the spray-drying process at 120 and 160 °C, the powders obtained from the Pr-E, Se-E, and Te-E emulsions indicated retention rates of 88.5 and 87.4%, 96.4 and 95.3%, and 96.8 and 96.0%, respectively. The astaxanthin retention values of the multilayer emulsions were higher compared to those provided by Shen and Quek [The results showed that the retention of astaxanthin from Se-E and Te-E dried at 120 and 160 °C incremented considerably  observed with respect to the air inlet temperatures of the dryer. This could be ascribed to the short exposure time of the emulsions during this spray-drying process. This technique has been used extensively in the food industry due to its short drying time and is therefore highly suitable for the dry heating of labile compounds [Moreover, there were no significant differences p > 0.0 observedompounds , the micFinally, the use of multilayer emulsions is preferable in spray drying processes with high temperatures, offering advantages such as greater stability of the emulsion and greater retention of the bioactive. Consequently, the use of this system could be an ideal option to produce dry emulsions, especially in the encapsulation of unstable bioactives such as astaxanthin.This investigation revealed that stable multilayer o/w emulsions that contain astaxanthin microencapsulated can be produced using LPI, CA, and CHI as ionic interfacial layers. All emulsions stabilized with one, two, and three interfacial layers  presented greater physical stability. However, the Se-E was the most stable compared to the Pr-E and Te-E when subjected to different pHs. Nevertheless, when the NaCl concentration was increased, no changes in the ζ-potential of any of the emulsions studied or destabilization were observed.Se-E and Te-E exhibited higher astaxanthin retention during spray drying in comparison to Pr-E. Finally, it can be stated that the results obtained in this research have given us a broader understanding of multilayer emulsion systems in the retention of astaxanthin for its potential application in the food industry.